Expression and Processing of a Bacterial Endoglucanase in Transgenic Mice

Zhang, Jun X; Meidinger, Roy G.; Forsberg, Cecil W.; Krell, Peter J.; Phillips, J. P.

doi:10.1006/abbi.1999.1243

Cited by 13 publications

(1 citation statement)

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“…Xylanases have been expressed and used as reporter proteins in bacteria and plants (Kamionka et al, 2004;Vickers et al, 2003). Bacterial family 10 xylanases (displaying activity on both, cellulose and hemicellulose) as well as true cellulases have been previously expressed in mammalian cells in order to improve nutritional performance of transgenic animals (Fontes et al, 1999;Soole et al, 1993;Zhang et al, 1997Zhang et al, , 1999. We have engineered B. subtilis xylanase A, a family 11 xylanase, which, in contrast to family 10 xylanases, exclusively catabolizes D-xylosecontaining substrates for optimal intracellular production as well as for high-level secretion in mammalian cells.…”

Section: Introductionmentioning

confidence: 99%

InXy and SeXy, compact heterologous reporter proteins for mammalian cells

Fluri

Kelm

Lesage

et al. 2007

Biotech & Bioengineering

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Mammalian reporter proteins are essential for gene-function analysis, drugscreening initiatives and as model product proteins for biopharmaceutical manufacturing. Bacillus subtilis can maintain its metabolism by secreting Xylanase A (XynA), which converts xylan into shorter xylose oligosaccharides. XynA is a family 11 xylanase monospecific for D-xylose containing substrates. Mammalian cells transgenic for constitutive expression of wild-type xynA showed substantial secretion of this prokaryotic enzyme. Deletion analysis confirmed that a prokaryotic signal sequence encoded within the first 81 nucleotides was compatible with the secretory pathway of mammalian cells. Codon optimization combined with elimination of the prokaryotic signal sequence resulted in an exclusively intracellular mammalian Xylanase A variant (InXy) while replacement by an immunoglobulin-derived secretion signal created an optimal secreted Xylanase A derivative (SeXy). A variety of chromogenic and fluorescence-based assays adapted for use with mammalian cells detected InXy and SeXy with high sensitivity and showed that both reporter proteins resisted repeated freeze/thaw cycles, remained active over wide temperature and pH ranges, were extremely stable in human serum stored at room temperature and could independently be quantified in samples also containing other prominent reporter proteins such as the human placental alkaline phosphatase (SEAP) and the Bacillus stearothermophilus-derived secreted alpha-amylase (SAMY). Glycoprofiling revealed that SeXy produced in mammalian cells was N- glycosylated at four different sites, mutation of which resulted in impaired secretion. SeXy was successfully expressed in a variety of mammalian cell lines and primary cells following transient transfection and transduction with adeno-associated virus particles (AAV) engineered for constitutive SeXy expression. Intramuscular injection of transgenic AAVs into mice showed significant SeXy levels in the bloodstream. InXy and SeXy are highly sensitive, compact and robust reporter proteins, fully compatible with pre-existing marker genes and can be assayed in high-throughput formats using very small sample volumes.

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